Tyrosine kinase inhibitors

ABSTRACT

The present invention relates to novel proteins that inhibit the activity of tyrosine kinases. In particular, the invention provides a tyrosine kinase inhibitor protein consisting of the cap region of a c-Abl protein. The invention also relates to the use of tyrosine kinase inhibitor proteins in the treatment and diagnosis of diseases, in particular cancers, in humans.

The present invention relates to novel proteins that inhibit theactivity of tyrosine kinases. The invention also relates to the use oftyrosine kinase inhibitor proteins in the treatment and diagnosis ofdiseases, in particular cancers, in humans.

All documents mentioned in the text and listed at the end of thedescription are incorporated herein by reference.

Protein tyrosine kinases are enzymes that transfer the terminalphosphate of adenosine triphosphate (ATP) to a specific tyrosine residueon a target protein. These enzymes are found in all multicellularorganisms and play a central role in the regulation of cellular growthand in the differentiation of complex eukaryotes.

There are two major classes of tyrosine kinases: transmembrane receptortyrosine kinases and non-receptor tyrosine kinases. Regulation of allprotein tyrosine kinases is essential for normal cellulardifferentiation and proliferation. While controlled activation oftyrosine kinases promotes normal proliferation, deregulated tyrosinekinases can cause neoplastic transformation. Examples from both classesof kinases have been shown to function as dominant oncogenes, generallyas a result of overexpression and/or structural alteration.

Transmembrane receptor tyrosine kinases are activated directly bybinding of peptide growth factors and cytokines to their extracellulardomains. Tyrosine kinases which fall within this class include receptorsfor platelet-derived growth factor, fibroblast growth factors,hepatocyte growth factor, insulin, insulin-like growth factor-1, nervegrowth factor, vascular endothelial growth factor and macrophage colonystimulating factor. The normal function of these receptors is to act astransducers of extracellular signals.

Some non-receptor tyrosine kinases are associated with cell surfacereceptors which do not have intrinsic tyrosine kinase activity. Forexample, members of the Src family of non-receptor protein tyrosinekinases in mammals (such as src, yes, fgr, fyn, lck, lyn, hck and blk)are mostly located on the cytoplasmic side of the plasma membrane, heldthere partly by their interaction with transmembrane receptors andpartly by covalently-attached lipid chains. These proteins are alsoinvolved in signal transduction pathways. However, not all non-receptorprotein tyrosine kinases are associated with transmembrane receptors.Some are found in the cytoplasm or even in the nucleus of cells. Therole of these proteins is in many cases unknown.

The c-Abl protein tyrosine kinase is another example of a non-receptortyrosine kinase. It was originally isolated as a cellular homologue ofthe v-abl oncogene of a transforming retrovirus, the Abelson murineleukaemia virus. The c-Abl protein is now known to be ubiquitouslyexpressed and highly conserved in metazoan evolution. The N-terminaldomain of the c-Abl protein, and the product of its paralog gene, Arg(ABL2), resemble Sic family kinases and consist of an SH3 domain, an SH2domain and a catalytic domain (reviewed in Superti-Purga andCourtneidge, 1995; Van Etten, 1999). However, in contrast to Src familykinases, c-Abl and Arg lack a short C-terminal regulatory-tail andinstead have a large C-terminal portion, encoded by a single exon andthus called the “last exon region”.

Although c-Abl was first identified 20 years ago, its physiological roleis still unclear. It has been implicated in a wide range of cellularprocesses including cell differentiation, cell division, cell adhesion,cell death and stress response (Van Etten, 1999; Wang, 2000). It isfound in both the nucleus and cytoplasm of cells and is thought toshuttle between the two subcellular departments (Taagepera et al.,1998). When expressed transiently in tissue culture cells, the abilityof c-Abl to become phosphorylated on tyrosine and phosphorylate cellularsubstrates is considerably weaker than that of the natural oncogenicform BCR-Abl or of c-Abl's viral counterpart, v-Abl, suggesting that itskinase activity is tightly regulated.

In humans, chronic myelogenous leukemia (CML) and a subset of acutelymphocytic leukemia (ALL) are causally linked to the presence of thePhiladelphia chromosome, which is the result of a translocation betweenchromosome 22 and chromosome 9. In this translocation, sequences of thefirst exon of the c-Abl tyrosine kinase gene (ABL1) are replaced bysequences from the BCR gene. Depending on the breakpoint in the BCRgene, the resulting fusion protein, BCR-Abl, can have molecular massesof 210, 190 (the two major forms) or 230 kDa The consequences of BCR-Ablon signal transduction pathways and the cellular effects have beenstudied extensively (Raitano et al., 1997). Dependent on the cell type,BCR-Abl expression results in enhanced proliferation, morphologicaltransformation, or abrogation of growth factor or adhesion dependence.In general, the effects are growth stimulatory and anti-apoptotic. Whenforced into the nuclei of cells, however, BCR-Abl induces apoptosis(Vigneri and Wang, 2001).

The common feature critical for all the biological effects of BCR-Abl isits constitutively high level of tyrosine kinase activity derived fromthe catalytic domain in its ABL1 moiety. A small molecular inhibitor ofBCR-Abl catalytic activity, STI571, has been described which binds tothe ATP-binding pocket of the catalytic domain, but also interacts withless conserved regulatory structural elements, affecting their function(Schindler et al., 2000). STI571 appears to target an Abl-specificinactive conformation of the catalytic domain, explaining its highselectivity for Abl over other tyrosine kinases. Although STI571initially appeared to be a promising therapeutic agent in clinicaltrials (Druker et al., 1996; Thiesing et al., 2000), it has recentlybeen found that patients treated with ST1517 relapse because of asecondary mutation in the ATP binding pocket (Gorre et al, 2001).

There is therefore a need for improved drugs capable of inhibitingBCR-Abl. Part of the difficulty in developing improved drugs to targetoncogenic forms of Abl is due to the fact that despite years ofinvestigation, the molecular mechanism responsible for regulation of thec-Abl tyrosine linase has remained elusive. In particular, the mechanismresponsible for natural inhibition of the cellular form of the enzyme isunknown.

Mutations have been identified in c-Abl, typically in the SH3 domain,that unleash the catalytic activity and often the oncogenic potential ofthe Abl protein. It is thought that these “deregulated” forms escape acritical mechanism that is responsible for the tight regulation of thewild-type protein (Pendergast et al., 1991; Mayer and Baltimore, 1994;reviewed in Van Etten, 1999). Many researchers have suggested that thesemutations enable c-Abl to escape regulation by a cellular inhibitor andseveral lines of evidence have contributed to this hypothesis. First,c-Abl and deregulated forms display equal levels of activity afterprecipitation or partial purification (Pendergast et al., 1991; Mayerand Baltimore, 1994; Dorey et al., 1999). Moreover, very high levels ofexpression in cells seem to exhaust regulation, as if through titrationof a cellular inhibitor (Pendergast et al., 1991). Expression in aheterologous system, such as in the yeast Schizosaccharomyces pombe,showed no difference in activity between c-Abl and deregulated forms,suggesting the absence of a vertebrate c-Abl inhibitor in fingi(Walkenhorst et al., 1996) and thus supporting this hypothesis. Otherelements, such as the identification of a considerable number ofproteins binding to the SH3 domain of Abl, have all contributed to thewide acceptance of a cellular inhibitor theory of c-Abl regulation(reviewed in Van Etten, 1999; Brasher and Van Etten, 2000).

There have been some suggestions that Abl might be regulated byintramolecular interactions. The crystal structure of regulated c-Src(reviewed in Sicheri and Kuriyan, 1997) prompted a mutational analysisthat supported the possibility of c-Abl being regulated byintramolecular interactions. It was suggested that these intramolecularinteractions would be similar to those identified in Src tyrosinekinases and would involve binding of the SH3 domain to the linkerbetween the SH2 and catalytic domains, and the catalytic domain itself(Barilá and Superti-Furga, 1998). However, researchers could notreconcile this theory that c-Abl could be autoinhibited with datashowing that c-Abl and deregulated forms of c-Abl had similar activityin vitro. The presence of a cellular inhibitor in the process of c-Ablregulation could not therefore be excluded and remains the dominantview. Moreover, the comparison to the regulation of Src family memberslacked explanations for the absence of a regulatory C-terminal tail inc-Abl (Barilá and Superti-Furga, 1998; Brasher and Van Etten, 2000; VanEtten, 1999).

In summary, despite having been the subject of extensive research formore than 20 years, the molecular mechanism by which c-Abl is regulatedand its role in the cell remain unknown. Protein tyrosine kinases suchas Abl play an essential role in the regulation of normal cellularproliferation and differentiation in multicellular organisms, asevidenced by the common incidence of mutations in genes encodingtyrosine kinase proteins in certain cancers. Given the importance oftyrosine kinase proteins in mammalian diseases and particularly incancer, there is an urgent need for a better understanding of the way inwhich medically important proteins, such as c-Abl, and the genesencoding them are regulated. An understanding of the way in whichtyrosine kinase proteins are regulated will lead to an understanding ofhow they become deregulated in disease, enabling effective inhibitors ofderegulated forms of protein tyrosine kinases to be developed for thetreatment of disease, in particular cancer.

SUMMARY OF THE INVENTION

The inventors have established that the tyrosine kinase activity ofc-Abl is regulated via autoinhibition. Contrary to the teaching in theprior art, regulation of the tyrosine kinase activity of c-Abl does notrequire an SH3 domain-dependent cellular inhibitor. A region of c-Ablfound at the N-terminus of the protein, herein described as a “capregion”, binds intramolecularly to c-Abl and is required to achieve andmaintain inhibition of tyrosine kinase activity. This cap region hasbeen found to be absent in all oncogenic forms of Abl and in particularin BCR-Abl resulting in deregulation and increased tyrosine kinaseactivity. However, it has been found that the cap region can reduce thetyrosine kinase activity of BCR-Abl forms and restore regulation,probably by binding directly to the Abl moiety of the oncogenic form.

Accordingly, the invention provides a tyrosine kinase inhibitor proteinconsisting of the cap region of a c-Abl protein, or a functionalequivalent thereof. The tyrosine kinase inhibitor proteins of theinvention are useful candidate agents for the development of drugs totreat disease caused by deregulated tyrosine kinase activity, as well asvaluable tools for research into the normal roles of tyrosine kinases incells.

The cap region has been shown to contain two conserved domains either orboth of which appear to be required for it to achieve and maintaininhibition of the tyrosine kinase activity of the c-Abl protein. Thefirst region, referred to herein as the “cap 1 domain”, is at least fouramino acids long and appears to be critical for binding the catalyticdomain. The second region, herein referred to as the “cap 6 domain”, isalso at least four amino acids long and appears to interact with the SH2and/or SH3 domains.

By a “cap region” of a c-Abl protein is meant a protein sequence ofbetween 20 and 250 amino acids derived from the N-terminal region of ac-Abl protein comprising a cap 1 domain and/or a cap 6 domain or afunctional equivalent thereof.

Where both the cap 1 domain and the cap 6 domain are present, theyshould be appropriately spaced so as to enable the cap 1 domain to bindthe catalytic domain of the target tyrosine kinase and the cap 6 domainto bind the SH2 and/or SH3 domains of the target tyrosine kinase. Thenumber of amino acids separating the cap 1 domain and the cap 6 domainmay vary provided that the cap 1 and cap 6 domains are spacedappropriately to enable them to bind to the target tyrosine kinase. Forexample, the cap 1 and cap 6 domains may be separated only by asufficient number of amino acids necessary to stretch between thecatalytic domain and the SH2 and/or SH3 domains. Alternatively, the cap1 and cap 6 domains may be separated by a larger number of amino acidswith the intervening amino acids “looping out” between the binding siteof the cap 1 domain and the binding site of the cap 6 domain.

Preferably, the cap 1 domain comprises the sequence KXXG, where X is Lor V. Preferably, the cap 6 domain comprises the sequence KENL.

Preferably, the cap region comprises additional conserved regionsoutside the cap 1 and cap 6 domains. Preferably, the cap regioncomprises a conserved stretch of amino acids upstream of the cap 6domain. Preferably, this conserved stretch of amino acids isapproximately 9 amino acids long and together with the cap 6 domainforms an alpha helix. Preferably, said conserved stretch of amino acidsupstream of the cap 6 domain comprises the amino acid sequenceLXEAZRWNS, where X is S or N and Z is A or R.

Preferably, the cap region consists of the N-terminal region of a c-Ablprotein and is encoded by the first exon and the first part of thesecond exon of this gene. More precisely, the cap region preferablyconsists of the N-terminal region of a c-Abl protein encoded by thefirst exon and the first 60 to 70 amino acids of the second exon. Thecap 1 domain is generally located in the first exon and the cap 6 domainis generally located in the second exon of the Abl gene.

In an even more preferred embodiment, the cap region may be derived fromthe N-terminal region of any c-Abl protein from any species including,but not limited to, vertebrates and invertebrates. Preferably, the capregion is derived from a mammalian c-Abl protein, preferably a humanc-Abl protein or a murine c-Abl protein. The c-Abl protein exists inhumans and in mice in two isoforms produced by splicing one of twoalternative first exons, type 1a and 1b, to a common second exon. Thetwo isoforms of c-Abl are referred to herein as Abl 1a and Abl 1b inhumans and Abl I and Abl IV in mice.

Where the cap region is derived from a human c-Abl protein, it may bederived from either human c-Abl 1a or human c-Abl 1b. Where the capregion is derived from human c-Abl 1a, it preferably consists of aminoacid residues 1-61 of the sequence of c-Abl 1a given in FIG. 8 or is afunctional equivalent thereof. Where the cap region is derived fromhuman c-Abl 1b, the cap region preferably consists of amino acidresidues 1-80 of the sequence of c-Abl 1a given in FIG. 8 or is afunctional equivalent thereof.

Where the cap region is derived from a murine c-Abl protein, it may bederived from either murine c-Abl I or murine c-Abl IV. Where the capregion is derived from murine c-Abl I, it preferably consists of aminoacids 1-63 of the sequence of c-Abl I as exemplified in FIG. 8 or is afunctional equivalent thereof. Where the cap region is derived frommurine c-Abl IV, it preferably consists of amino acids 1-80 of c-Abl IVas exemplified in FIG. 8 or is a functional equivalent thereof.

The precise boundaries for the cap region given above are, of course,approximate, in that proteins that have been truncated within orextended beyond these boundaries may still be effective as tyrosinekinase inhibitor proteins, providing that the cap 1 and cap 6 domainshave not been deleted. Furthermore, as and when new c-Abl proteins areidentified and characterised, the cap regions from these proteins may beused as tyrosine kinase inhibitor proteins according to the invention.

The cap region of c-Abl has been shown to auto-inhibit c-Abl duringnormal regulation without any requirement for a cellular inhibitor andto restore regulation of tyrosine kinase activity to oncogenic forms ofAbl lacking the cap region. Although the Applicant does not wish to bebound by this theory, it is believed that the cap region may alsoinhibit the tyrosine kinase activity of proteins other than c-Abl. Thetyrosine kinase inhibitor proteins of the invention may thereforeinhibit any tyrosine kinase protein containing SH2 and SH3 domains.Preferably, the tyrosine kinase inhibitor proteins and functionalequivalents of the invention inhibit Abl proteins, preferably oncogenicforms of Abl, including, for example, BCR-Abl. Other tyrosine kinaseproteins which may be inhibited by the tyrosine kinase inhibitorproteins and functional equivalents of the invention include members ofthe src family, such as src, fyn, yes, lyn, lck, blk, hck, fgr and yrk.Other examples of tyrosine kinase proteins which may be inhibited by thetyrosine kinase inhibitor proteins and functional equivalents of theinvention include, frk, brk, srm, sad, btk, itk, tec, mkk2, txk, csk,ctk, zap70, syk, fes/fps and fer. Preferably, the oncogenic forms ofthese proteins are inhibited by the tyrosine kinase inhibitor proteinsof the invention or functional equivalents thereof. Examples ofoncogenic forms of tyrosine kinase proteins which may be inhibited bythe tyrosine kinase inhibitor proteins and functional equivalents of theinvention include v-Src and v-Fyn.

Functional equivalents of the tyrosine kinase inhibitor proteinsdescribed above are also included within the scope of the invention. Theterm “functional equivalent” is used herein to describe variants,derivatives and homologues of the tyrosine kinase inhibitor proteins ofthe invention that retain the ability to inhibit tyrosine kinaseproteins.

Functional equivalents of the tyrosine kinase inihibitor proteins of theinvention thus include natural biological variants (e.g. allelicvariants or geographical variants within the species from which thetyrosine kinase inhibitor proteins are derived). Functionally-equivalentvariants of the tyrosine kinase inhibitor proteins of the invention alsoinclude, for example, mutants containing amino acid substitutions,insertions or deletions from the wild type protein sequences presentedherein. Such mutants may include polypeptides in which one or more ofthe amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code. Typical such substitutions are among Ala,Val, Leu and Be; among Ser and Thr, among the acidic residues Asp andGlu; among Asn and Gln; among the basic residues Lys and Arg; or amongthe aromatic residues Phe and Tyr. Particularly preferred are variantsin which several, i.e. between 5 and 10, 1 and 5, 1 and 3, 1 and 2 orjust 1 amino acids are substituted, deleted or added in any combination.Especially preferred are silent substitutions, additions and deletions,which do not alter the properties and activities of the protein. Alsoespecially preferred in this regard are conservative substitutions.

Functionally-equivalent variants with improved function from that of thewild type sequences may also be designed through the systematic ordirected mutation of specific residues in the protein sequence.Improvements in function that may be desired will include improvementssuch as a greater ability to inhibit tyrosine kinase activity.

The term “functional equivalent” also refers to molecules that arestructurally similar to the tyrosine kinase inhibitor proteins of theinvention or that contain similar or identical tertiary structure. Suchfunctional equivalents may be derived from the natural tyrosine kinaseinhibitor proteins or they may be prepared synthetically or usingtechniques of genetic engineering. In particular, synthetic moleculesthat are designed to mimic the tertiary structure or active site of thenaturally-occurring tyrosine kinase inhibitor proteins of the inventionare considered to be functional equivalents. In particular, syntheticmolecules which are designed to mimic the tertiary structure of the cap1 domain and the cap 6 domain in naturally-occurring cap regions areconsidered to be functional equivalents. Where it is desired that boththe cap 1 domain and the cap 6 domain be present, the skilled personwill readily be able to design molecular mimetics of cap regions fromnatural c-Abl proteins which contain the cap 1 and cap 6 domains in thecorrect spatial conformation to bind and inhibit a target tyrosinekinase.

The term “functional equivalent” also includes derivatives of thetyrosine kinase inhibitor proteins of the invention. By the term“derivative” is meant tyrosine kinase inhibitor proteins that have beenmodified, for example, by the covalent attachment of non-protein groups.In particular, the term “derivative” includes tyrosine kinase inhibitorproteins of the invention which have undergone post-translationmodification. Post-translation modification of c-Abl appears to play arole in controlling its activity (Jackson & Baltimore, 1989). It istherefore postulated that the function of the tyrosine kinase inhibitorproteins of the invention may be affected by post-translationmodification and in particular, the covalent attachment of a fatty aclygroup or a prenyl group. Preferably, the post-translation modificationinvolves covalent attachment of a fatty acyl group or a prenyl group tothe tyrosine kinase inhibitor protein. Preferably, derivatives of thetyrosine kinase inhibitor proteins of the invention includemyristoylated and palmitoylated derivatives.

The term “functional equivalent” also refers to homologues of thetyrosine kinase inhibitor proteins of the invention. By “homologue” ismeant a protein exhibiting a high degree of similarity or identity tothe amino acid sequence of the tyrosine kinase inhibitor proteins of theinvention. By “similarity” is meant that, at any particular position inthe aligned sequences, the amino acid residue is of a similar typebetween the sequences. By “identity” is meant that at any particularposition in the aligned sequences, the amino acid residue is identicalbetween the sequences.

Preferably, homologues possess greater than 40% identity with thenaturally-occurring tyrosine kinase inhibitor protein sequences. Morepreferably, homologues according to the invention show greater than 50%,60%, 70%, 80%, 90%, 95%, 97%, 98% or 99% sequence identity with thesequence of the natural protein. Percentage identity, as referred toherein, is as determined using BLAST version 2.1.3 using the defaultparameters specified by the NCBI (the National Center for BiotechnologyInformation; http://www.ncbi.nlm.nih.gov/) [Blosum 62 matrix; gap openpenalty-11 and gap extension penalty=1]. Tools such as PROSITE(http://expasy.hcuge.ch/sprot/prosite.html), PRINTShttp://iupab.leeds.ac.uklbmb5dp/prints.html), Profiles(http://ulrec3.unil.ch/software/PFSCAN_form.html), Pfam(http://www.sanger.ac.uk/software/pfam), Identify(http://dna.stanford.edu/identify/) and Blocks(http://www.blocks.fhcrc.org) databases may also be used to identifyhomologues, as well as hidden Markov models (HMMs; preferably profileHMMs). Such homologues may include proteins in which one or more of theamino acid residues are substituted with another amino acid residueprovided that the function of the protein is retained as a tyrosinekinase inhibitor. Any such substituted amino acid residue may or may notbe a naturally occurring amino acid.

In particular, functional equivalents of the tyrosine kinase inhibitorproteins of the invention include cap regions derived from the humanparalog of c-Abl, Arg. Preferably, the cap regions derived from Argconsist of the amino acid sequences for the cap regions of Arg 1a andArg 1b given in FIG. 8.

The term “functional equivalents” also includes active fragments of thenaturally-occurring tyrosine kinase inhibitor proteins, variants,derivatives and homologues described above. By “active fragment” ismeant any fragment of the tyrosine kinase inhibitor proteins, variants,derivatives or homologues that retain the ability to act as tyrosinekinase inhibitors. Such fragments may include both a cap 1 domain and acap 6 domain or may include only a cap 1 domain or only a cap 6 domain.

According to a further aspect of the invention, there is provided afusion protein comprising a tyrosine kinase inhibitor protein of theinvention or functional equivalent thereof, as described in any one ofthe embodiments recited above. The tyrosine kinase inhibitor protein orfunctional equivalent may be genetically or chemically fused to one ormore peptides or polypeptides. Preferably, the tyrosine kinase inhibitorprotein or functional equivalent is fused to a marker domain.Preferably, the marker domain is a fluorescent tag, an epitope tag thatallows purification by affinity binding, an enzyme tag that allowshistochemical or fluorescent labelling, or a radiochemical tag. In oneembodiment, the fluorescent tag is a green fluorescent protein (GFP) ora fluorescent derivative thereof such as YFP or CFP (see Prasher et al,(1995), Trends in Genetics, 11(8), 320).

Such fusion proteins will be useful in a variety of methods forestablishing the role of the tyrosine kinase inhibitor proteins of theinvention. For example, they can be used to facilitate the detection ofthe tyrosine kinase inhibitor proteins of the invention.

Methods for the generation of fusion proteins are standard in the artand will be known to the skilled reader. For example, most generalmolecular biology, microbiology recombinant DNA technology andimmunological techniques can be found in Sambrook et al., (MolecularCloning, A Laboratory Manual, Cold Harbor-Laboratory Press, Cold SpringHarbor, N.Y., 2000) or Ausubel et al., (Current Protocols in MolecularBiology, Wiley Interscience, New York, 1991).

Generally, fusion proteins may be most conveniently generatedrecombinantly from nucleic acid molecules in which two nucleic acidsequences are fused together in frame. These fusion proteins will beencoded by nucleic acid molecules that contain the relevant codingsequence of the fusion protein in question.

The tyrosine kinase inhibitor proteins, functional equivalents andfusion proteins of the invention may be prepared in recombinant form byexpression in a host cell. Suitable expression methods are well known tothose of skill in the art and many are described in detail by SambrookJ. et al Molecular cloning: a laboratory manual New York: Cold SpringHarbour Laboratory Press, 2000) and Fernandez J. M. & Hoeffler J. P.(Gene expression systems. Using nature for the art of expression ed.Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo,Toronto, 1998.) The proteins and functional equivalents of the presentinvention can also be prepared using conventional techniques of proteinchemistry, for example by chemical synthesis.

According to a further embodiment, the invention provides antibodiesthat bind to a tyrosine kinase inhibitor protein or functionalequivalent, as described above. The invention further providesantibodies that bind to a complex of a tyrosine kinase inhibitor proteinand a deregulated tyrosine kinase protein. Antisera and monoclonalantibodies can be made by standard protocols using the tyrosine kinaseinhibitor protein or functional equivalent as an immunogen (see, forexample, Antibodies: A Laboratory Manual ed. By Harlow and Lane, ColdSpring Harbor Press, 1988). As used herein, the term “antibody” includesfragments of antibodies that also bind specifically to a tyrosine kinaseinhibitor protein or functional equivalent thereof. The term “antibody”further includes chimeric and humanised antibody molecules havingspecificity for the tyrosine kinase inhibitor proteins of the inventionand for functional equivalents thereof. Antibodies that bind to tyrosinekinase inhibitor proteins are useful in a variety of methods forelucidating the function of tyrosine kinase proteins and in particularderegulated tyrosine kinase proteins. For example, they can be used todemonstrate the presence of a tyrosine kinase inhibitor protein bound toa tyrosine kinase protein. They can also be used to measure the quantityof a tyrosine kinase inhibitor protein in a cell extract. In some cases,it will be desirable to attach a label group to the antibody in order tofacilitate detection. Preferably, the label is an enzyme, a radiolabelor a fluorescent tag. These antibodies may also have medicalapplications. In particular, an antibody that binds to a complex of atyrosine kinase inhibitor protein bound to a deregulated tyrosine kinasemay act to keep the complex associated, ensuring that the normaltyrosine kinase activity of the protein is restored, reducing itsoncogenic capabilities.

The invention also provides a nucleic acid molecule encoding a tyrosinekinase inhibitor protein, functional equivalent thereof, or fusionprotein as described above. Specific examples of nucleic acid moleculesencoding tyrosine kinase inhibitor proteins according to the inventioninclude those nucleic acid sequences presented in FIG. 9 herein.However, included in this aspect of the invention are nucleic acidmolecules that are at least 30%, 40%, 50%, 60% or 70% identical, morepreferably, at least 80%, 90%, 95%, 98% or 99% identical over theirentire length to a nucleic acid molecule encoding a tyrosine kinaseinhibitor protein according to any one of the embodiments of theinvention described above. Such nucleic acid molecules include single-or double-stranded DNA, cDNA and RNA, as well as synthetic nucleic acidspecies. Preferably, the nucleic acid molecules are DNA or cDNAmolecules. According to one embodiment, the invention provides a nucleicacid molecule including a nucleic acid sequence presented in FIG. 9.

The nucleic acid sequences will have medical applications. For example,a nucleic acid sequence encoding a tyrosine kinase protein of theinvention may be supplied to a patient instead of supplying the tyrosinekinase inhibitor protein directly to the patient. In addition, it hasbeen shown that regulation of a deregulated tyrosine kinase proteinlacking the cap region, BCR-Abl, can be restored by integrating anucleic acid sequence encoding the cap region protein into the geneencoding the tyrosine kinase protein. These results demonstrate thatnucleic acid molecules encoding the tyrosine kinase inhibitor proteinsof the invention have potential applications in gene therapy.

The invention also includes cloning and expression vectors incorporatingthe nucleic acid molecules. Such expression vectors may additionallyincorporate regulatory sequences such as enhancers, promoters, ribosomebinding sites and termination signals in the 5′ and 3′ untranslatedregions of genes, that are required to ensure that the coding sequenceis properly transcribed and translated, or to regulate the expression ofthe protein relative to the growth of the cell in which it is expressed.Also, control sequences may be included that encode signal peptides orleader sequences. These leader or control sequences may be removed bythe host during post-translational processing. In some cases, thevectors may incorporate signal sequences which direct the expressedproteins to specific subcellular locations. For example, it may bedesirable to assess the effect of a tyrosine kinase inhibitor protein inthe nucleus, in which case a nuclear localisation signal may be includedin the vector.

Vectors according to the invention include plasmids and viruses(including both bacteriophage and eukaryotic viruses), as well as otherlinear or circular DNA carriers, such as those employing transposableelements or homologous recombination technology. Many such vectors andexpression systems are known and documented in the art (see, forexample, Fernandez J. M. & Hoeffler J. P. in Gene expression systems.Using nature for the art of expression ed. Academic Press, San Diego,London, Boston, New York, Sydney, Tokyo, Toronto, 1998). Suitable viralvectors include baculovirus-, adenovirus- and vaccinia virus-basedvectors.

Suitable hosts for recombinant expression include commonly usedprokaryotic species, such as E. coli, or eukaryotic yeasts that can bemade to express high levels of recombinant proteins and that can easilybe grown in large quantities. Mammalian cell lines grown in vitro arealso suitable, particularly when using virus-derived expression systems.Another suitable expression system is the baculovirus expression systemthat involves the use of insect cells as hosts. An expression system mayalso constitute host cells that have the appropriate encoding nucleicacid molecules incorporated into their genome. Proteins may also beexpressed in vivo, for example, in insect larvae or in mammaliantissues.

A variety of techniques may be used to introduce the vectors accordingto the present invention into prokaryotic or eukaryotic host cells.Suitable transformation or transfection techniques are well described inthe literature (see, for example, Sambrook et al, Molecular cloning: alaboratory manual New York: Cold Spring Harbour Laboratory Press, 2000;Ausubel et al, Current Protocols in Molecular Biology, WileyInterscience, New York, 1991; Spector, Goldman & Leinwald, Spector et alCells, a laboratory manual; Cold Spring Harbour Laboratory Press, 1998).In eukaryotic cells, expression systems may either be transient (e.g.episomal) or permanent (such as by chromosomal integration) according tothe needs of the system.

The invention also provides antisense nucleic acid molecules whichhybridise under high stringency hybridisation conditions to the nucleicacid molecules encoding the tyrosine kinase inhibitor proteins orfunctional equivalents thereof. High stringency hybridisation conditionsare defined herein as overnight incubation at 42° C. in a solutioncomprising 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH7.6), 5× Denhardts solution, 10% dextransulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 0.1×SSC at approximately 65° C.

In a preferred embodiment of this aspect of the invention, a labelcapable of being detected is attached to these antisense nucleic acidmolecules. Preferably, the label is selected from the group consistingof radioisotopes, fluorescent compounds and enzymes.

These antisense nucleic acid molecules may be used as probes to detectdefects in the nucleic acid molecules encoding a tyrosine kinase proteinto which the tyrosine kinase inhibitor protein of the invention binds.The antisense nucleic acid molecules of the invention may therefore beuseful in diagnostic assays. For example, it has been shown that the capregion is generally missing in oncogenic forms of the tyrosine kinaseprotein Abl, such as BCR-Abl but that reintroduction of a nucleic acidmolecule encoding the cap region restores regulation. The antisensenucleic acid molecules of the invention may therefore be useful fordetecting whether a cap region is present in the nucleic acid moleculeencoding Abl and to screen for mutation in the cap region of an Ablprotein. The antisense nucleic acid molecules can also be used to detectwhether a nucleic acid encoding a cap region has been successfullyintroduced into a gene encoding an oncogenic form of Abl, such asBCR-Abl.

The invention also includes transformed or transfected prokaryotic oreukaryotic host cells containing a nucleic acid molecule encoding atyrosine kinase inhibitor protein or functional equivalent thereof asdescribed above or an antisense nucleic acid molecule which hybridisesto such a nucleic acid molecule.

A further aspect of the invention provides a method for preparing atyrosine kinase inhibitor protein or a functional equivalent thereof ora fusion protein as defined above, which comprises culturing a host cellcontaining a nucleic acid molecule encoding a tyrosine kinase inhibitorprotein or functional equivalent thereof or a fusion protein accordingto the invention under conditions whereby said protein is expressed andrecovering said protein thus produced.

As the skilled reader will appreciate, the identification of thetyrosine kinase inhibitor proteins of the present invention and, inparticular, the identification of the role of such proteins in theautoinhibition of c-Abl will result in an increased understanding of theway in which tyrosine kinase proteins become deregulated in diseasessuch as cancer. Although the applicant does not wish to be bound by anyspecific theory, it is considered possible that the normalautoinhibition of c-Abl by its N-terminal cap region may be disrupted bymolecules that prevent the cap region from interacting with thecatalytic and SH2 and/or SH3 domains. Such activator compounds willincrease the tyrosine kinase activity of c-Abl and may be useful targetsfor the development of drugs. Once such activator compounds have beenidentified, it may be possible to identify modulator compounds thatinhibit the ability of the activator compounds to disrupt normalautoinhibition of c-Abl by its N-terminal cap region.

A further aspect of the invention therefore provides a method ofidentifying an activator compound that inhibits autoinhibition of c-Ablby the cap region comprising contacting c-Abl with a candidate activatorcompound and assessing whether binding between the cap region of c-Abland the catalytic and SH2 and/or SH3 domains of c-Abl has beeninhibited.

There are numerous methods of determining if the candidate activatorcompounds binds at a site on the c-Abl protein that prevents bindingbetween the cap region and the catalytic domain and the SH2 and/or SH3domains. In particular, competition assays can be used to determine if acandidate activator compound prevents autoinihibition by the cap region.

In order to identify candidate activator compounds of the presentinvention, both the cap region of c-Abl and c-Abl proteins lacking thecap region can be used to screen libraries of compounds in any of avariety of drug screening techniques.

Candidate activator compounds may be isolated from, for example, cells,cell-free preparations, chemical libraries, or natural product mixtures.Suitable compounds might include polypeptides, such as enzymes,receptors, antibodies, and structural or functional mimetics of thesepolypeptides, including peptides and peptidomimetics. These may eitherbe natural compounds, isolated from natural sources, or may be syntheticor recombinant.

The c-Abl cap region that is employed in such a screening technique maybe free in solution, affixed to a solid support, borne on a cellsurface, or located intracellularly. The adherence of a candidateactivator compound to a surface bearing the c-Abl cap region can bedetected by means of a label directly or indirectly associated with thecandidate activator compound or in an assay involving competition with alabelled competitor. In general, such screening procedures may involveusing appropriate cells or cell membranes that express the appropriateprotein, that are then contacted with a candidate activator protein toobserve binding. Binding may be detected, for example, using a yeast2-hybrid screen.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest (for example, see International patentapplication WO84/03564). In this method, large numbers of differentsmall test compounds are synthesised on a solid substrate, which maythen be reacted with the c-Abl cap region and washed. One way ofimmobilising the target protein is to use non-neutralising antibodies.Bound protein may then be detected using methods that are well known inthe art, including using biophysical techniques such as surface plasmonresonance and spectroscopy. Purified protein can also be coated directlyonto plates for use in the aforementioned drug screening techniques.

In a preferred embodiment, a competition assay is performed wherein thec-Abl cap region or a c-Abl protein lacking the cap region is attachedto a solid support. Any suitable solid support may be used, such as, forexample, sepharose beads. Methods for linking proteins to solid supportsare well known to those skilled in the art.

For example, when the c-Abl cap region is attached to a solid support,the detection of binding of a c-Abl protein lacking the cap region caneasily be achieved, for example using a labelled antibody specific forthe SH2 domain or the SH3 domain. Alternatively, a c-Abl protein lackinga cap region may be attached to the solid support and detection of thebinding of the cap region can be achieved using a labelled antibodyspecific for the cap region. Suitable labels include: enzymes, such ashorseradish peroxidase (HRP) and chloramphenicol acetyl transferase(CAT); digoxygenin (DIG); fluorescein; and radioisotopes such as ¹²⁵I,³H and ¹⁴C.

Once a candidate activator compound has been identified using the assayof the present invention, it will be desirable to test the activatorcompound in a cell containing c-Abl in order to determine its effect onthe tyrosine kinase activity of c-Abl.

The invention further provides a method for identifying a modulatorcompound that restores autoinhibition of c-Abl by the cap regioncomprising contacting c-Abl and an activator compound, as describedabove, with a candidate modulator compound and assessing whether bindingbetween the cap region of c-Abl and the catalytic and SH2 and/or SH3domains of c-Abl is restored.

Candidate modulator compounds may be identified by conducting screeningassays to identify compounds that interact with the activator compounds.The ability of a modulator compound to compete with an activatorcompound can then be assessed using any method known to the personskilled in the art. In particular, competitive assays such as thosedescribed above may be carried out in the presence of an activatorcompound and then in the presence of an activator compound and amodulator compound. Preferably, the step of assessing whether bindingbetween the cap region and the catalytic domain and the SH2 and/or SH3domains of c-Abl has been restored comprises detecting the restorationof normal (low) tyrosine kinase activity.

The invention further provides a compound that is an activator compoundor a modulator compound, identified or identifiable by the screeningmethod mentioned above. It will be understood that modulators of theinvention are not limited to those identified by the above method butinclude any activator compounds which activate c-Abl by inhibitingbinding between the cap region of c-Abl and the catalytic and SH2 and/orSH3 domains, as well as any modulator compounds that restore binding byinhibiting an activator compound.

The identification of the function of the cap region of c-Abl protein asan inhibitor of tyrosine kinase activity allows tyrosine kinase proteinsto be regulated in vivo, for example, as some form of therapy, or invitro, for example, to modulate the activity of protein tyrosineactivity in tissue culture. According to one aspect of this embodimentof the invention, there is thus provided a method of modulating theactivity of a tyrosine kinase protein comprising providing a cell with atyrosine kinase inhibitor protein or a functional equivalent thereof, anucleic acid molecule encoding a tyrosine kinase inhibitor protein, anantisense nucleic acid molecule that binds to a nucleic acid moleculeencoding a tyrosine kinase protein, an activator compound or a modulatorcompound as described above. Preferably, the target protein tyrosinekinase is an oncogenic tyrosine kinase protein, preferably an oncogenicform of Abl, preferably BCR-Abl.

According to a further aspect of the invention, there is provided theuse of the cap region of a c-Abl protein, as defined above, as atyrosine kinase inhibitor.

The ability to modulate tyrosine kinase activity in cells and inparticular to decrease the tyrosine kinase activity of oncogenictyrosine kinase proteins will be a useful tool for researchers seekingto understand how deregulation of tyrosine kinase proteins results indiseases such as cancer.

As referred to above, the tyrosine kinase inhibitor proteins, functionalequivalents and modulators of the invention have a wide variety ofpotential medical applications. In particular, the tyrosine kinaseinhibitor proteins of the invention have applications in the treatmentof diseases associated with deregulated tyrosine kinase activity sincethey are able to restore regulated tyrosine kinase activity.

Nucleic acids encoding the tyrosine kinase inhibitor proteins of theinvention or functional equivalents thereof may also be introduced totreat such diseases. The nucleic acid molecules may be used to expressthe tyrosine kinase inhibitor protein in a target cell. Alternatively, aspecific embodiment of the invention involves integrating the nucleicacid encoding the tyrosine linase inhibitor protein into a gene encodinga deregulated tyrosine kinase protein which has no cap region.Preferably, the tyrosine kinase may be a deregulated form of c-Abl,preferably BCR-ABL.

Accordingly, the invention further provides a tyrosine kinase inhibitorprotein or a functional equivalent thereof, a nucleic acid moleculeencoding said tyrosine kinase inhibitor protein or functionalequivalent, an antisense nucleic acid molecule, an activator compound ora modulator compound as described above, for use as a pharmaceutical.

A further aspect of the invention includes a pharmaceutical compositioncomprising a tyrosine kinase inhibitor protein or a functionalequivalent thereof, a nucleic acid molecule encoding said tyrosinekinase inhibitor protein or functional equivalent, an antisense nucleicacid molecule, an activator compound or a modulator compound accordingto any one of the embodiments of the invention recited above, inconjunction with a pharmaceutically-acceptable carrier molecule.

Carrier molecules may be genes, polypeptides, antibodies, liposomes,polysaccharides, polylactic acids, polyglycolic acids and inactive virusparticles or indeed any other agent provided that the carrier does notitself induce toxicity effects or cause the production of antibodiesthat are harmful to the individual receiving the pharmaceuticalcomposition. Carriers may also include pharmaceutically acceptable saltssuch as mineral acid salts (for example, hydrochlorides, hydrobromides,phosphates, sulphates) or the salts of organic acids (for example,acetates, propionates, malonates, benzoates). Pharmaceuticallyacceptable carriers may additionally contain liquids such as water,saline, glycerol, ethanol or auxiliary substances such as wetting oremulsifying agents, pH buffering substances and the like. Carriers mayenable the pharmaceutical compositions to be formulated into tablets,pills, dragees, capsules, liquids, gels, syrups, slurries, suspensionsto aid intake by the patient. A thorough discussion of pharmaceuticallyacceptable carriers is available in Remington's Pharmaceutical Sciences(Mack Pub. Co., N.J. 1991).

The amount of the active compound in the composition should also be in atherapeutically-effective amount. The phrase “therapeutically effectiveamount” used herein refers to the amount of agent needed to treat orameliorate a targeted disease or condition. An effective initial methodto determine a “therapeutically effective amount” may be by carrying outassays in the transgenic organism model, although more accurate testsmust be carried out on the target organism if initial tests aresuccessful. The transgenic organism model may also yield relevantinformation such as the preferred routes of administration that willlead to maximum effectiveness. The exact therapeutically-effectivedosage will generally be dependent on the patient's status at the timeof administration. Factors that may be taken into consideration whendetermining dosage include the severity of the disease state in thepatient, the general health of the patient, the age, weight, gender,diet, time and frequency of administration, drug combinations, reactionsensitivities and the patient's tolerance or response to the therapy.The precise amount can be determined by routine experimentation but mayultimately lie with the judgement of the clinician. Generally, aneffective dose will be from 0.01 mg/kg (mass of drug compared to mass ofpatient) to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositionsmay be administered individually to a patient or may be administered incombination with other agents, drugs or hormones.

Uptake of a pharmaceutical composition by a patient may be initiated bya variety of methods including, but not limited to enteral,intra-arterial, intrathecal, intramedullary, intramuscular, intranasal,intraperitoneal, intravaginal, intravenous, intraventricular, oral,rectal (for example, in the form of suppositories), subcutaneous,sublingual, transcutaneous applications (for example, see WO98/20734) ortransdermal means. Gene guns or hyposprays may also be used toadminister pharmaceutical compositions. Typically, however, thetherapeutic compositions may be prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection may also beprepared. Direct delivery of the compositions can generally beaccomplished by injection, subcutaneously, intraperitoneally,intravenously or intramuscularly, or delivered to the interstitial spaceof a tissue. The compositions can also be administered into a lesion.Dosage treatment may be a single dose schedule or a multiple doseschedule.

The invention also includes the use of a tyrosine kinase inhibitorprotein or a functional equivalent thereof, a nucleic acid moleculeencoding said tyrosine kinase inhibitor protein or functional equivalentthereof, an antisense nucleic acid molecule, an activator compound or amodulator compound as described above, in the manufacture of amedicament for treating a disease or a condition associated withaberrant tyrosine kinase activity.

The tyrosine kinase inhibitor proteins and functional equivalents, thenucleic acid molecules encoding these tyrosine kinase inhibitor proteinsand functional equivalents, the antisense nucleic acid molecules, theactivator compounds and the modulator compounds of the invention will beuseful in the manufacture of medicaments for treating diseasesassociated with aberrant tyrosine kinase activity.

The tyrosine kinase inhibitor proteins and functional equivalents, thenucleic acid molecules encoding these tyrosine kinase inhibitor proteinsand functional equivalents, and the modulator compounds of the inventionwill be useful in the manufacture of medicaments for treating diseasesassociated with an aberrantly high level of tyrosine kinase activity.Preferably, such a disease is cancer, more preferably leukaemia Theantisense nucleic acid molecules and activator compounds of theinvention will be useful in the manufacture of medicaments for treatingdiseases associated with an aberrantly low level of tyrosine kinaseactivity, such as agammaglobulinemia. The tyrosine kinase inhibitorproteins and functional equivalents, the nucleic acid molecules encodingthese tyrosine kinase inhibitor proteins and functional equivalents, theantisense nucleic acid molecules, the activator compounds and themodulator compounds of the invention will also be useful in thetreatment of neurological disorders caused by aberrant tyrosine kinaseactivity.

According to a still further aspect of the invention, there is provideda method of treating a disorder or disease associated with aberranttyrosine kinase activity in a patient, comprising administering to thepatient a tyrosine kinase inhibitor protein or functional equivalentthereof, a nucleic acid molecule encoding said tyrosine kinase inhibitorprotein or functional equivalent, an antisense nucleic acid molecule, anactivator compound, a modulator compound or a composition as describedabove in a therapeutically-effective amount. Preferred patients aremammals, more preferably humans. Preferably, the disorder or diseaseassociated with aberrant tyrosine kinase activity is a neurologicaldisorder, agammaglobulinemia or cancer, preferably leukaemia.

The invention further comprises a method of diagnosing a conditionassociated with an aberrant activity of a tyrosine kinase proteincomprising measuring the level of an aberrant tyrosine kinase protein ina cell sample obtained from a patient using a tyrosine kinase inhibitorprotein of the invention. As indicated above, the tyrosine kinaseinhibitor proteins of the invention are supposed to inhibit theoncogenic forms of tyrosine kinase proteins but not the cellular forms.For example, the N-terminal cap region of c-Abl can inhibit BCR-Abl,probably by binding to it. In contrast, the N-terminal cap region ofc-Abl does not bind c-Abl, presumably because the binding site isalready occupied by the endogenous c-Abl cap region. Accordingly, thepresence of an oncogenic tyrosine kinase may be diagnosed by detectingthe presence and the level of complexes comprising a tyrosine kinaseinhibitor protein of the invention, bound to an oncogenic tyrosinekinase. Preferably, the level of the complex in the sample is measuredusing antibodies against the tyrosine kinase inhibitor protein or thecomplex, as described previously.

The invention further comprises a method of diagnosing a conditionassociated with an aberrant activity of a tyrosine kinase proteincomprising using a nucleic acid molecule or an antisense nucleic acidmolecule, as described above, to screen for mutations in the cap regionof the protein tyrosine kinase Abl.

In another embodiment of the invention, a nucleic acid molecule asdescribed above may be used to create a transgenic animal, mostconunonly a rodent. The modification of the animal's genome may eitherbe done locally, by modification of somatic cells or by germ linetherapy to incorporate inheritable modifications. Such transgenicanimals may be particularly useful in assessing whether integration ofnucleic acid molecules encoding tyrosine kinase inhibitor proteins ofthe invention can be used to restore regulation of tyrosine kinase in ananimal model in which the tyrosine kinase activity has been deregulated,causing cancer. The transgenic animals will also be useful in assessingthe effectiveness of modulator compounds, as described above.

As indicated above, the tyrosine kinase inhibitor proteins of theinvention will be useful in developing a more detailed understanding ofthe role of tyrosine kinases and the way in which they are regulated.The tyrosine kinase inhibitor proteins of the invention are the resultof the initial discovery by the inventors that the cap region of c-Ablis involved in its auto-inhibition. The inventors have also developed atechnique for assessing the effects of deregulating c-Abl in particular.Specifically, they have developed a c-Abl protein that contains amutation that introduces a Tobacco-Etch-Virus (TEV) protease cleavagesite roughly at the boundary between the cap region and the SH3 domain.The mutation does not affect the normal function of c-Abl but treatmentof cells expressing this protein with TEV protease results in cleavageof the cap region and deregulation of the tyrosine kinase activity ofc-Abl in vitro. This TEV construct hence has clear applications in thestudy of the effects of the activation of c-Abl activity.

Accordingly, according to a further aspect of the invention, there isprovided a c-Abl protein comprising a protease cleavage site locatednear the boundary of the cap region and the SH3 domain. Preferably, saidprotease cleavage site is a TEV protease cleavage site. Where the c-Ablprotein is a human type 1b c-Abl protein, the cleavage site ispreferably located at residues 82-85, such that cleavage occurs atresidue 78, cleaving residues 1-77 of the N-terminal domain.

According to a further aspect of the invention, there is provided afusion protein comprising a c-Abl protein comprising a protease cleavagesite bound to a marker domain, as described above in respect of thetyrosine inhibitor proteins in general.

According to a further aspect of the invention, there is provided amethod for activating the tyrosine kinase activity of c-Abl in a cellcomprising supplying the cell with a c-Abl protein comprising a proteasecleavage site as described above and supplying the cell with a protease.Preferably, the method further comprises removing the cleaved N-terminaldomain to ensure that the activated c-Abl protein is not inhibited bybinding of the cleaved N-terminal domain to it.

There is also provided a method for producing an activated c-Abl proteincomprising cleaving a c-Abl protein comprising a protease cleavage site,as described above, with a protease and isolating the cleaved C-terminalregion of the c-Abl protein.

There is also provided a method of producing a tyrosine kinase inhibitorprotein according to the invention comprising cleaving a c-Abl proteincomprising a protease site as described above with a protease andisolating the cleaved N-terminal cap region of the c-Abl protein.

The invention also provides nucleic acid molecules encoding the c-Ablproteins and fusion proteins comprising a protease domain as describedabove, as well as vectors comprising the nucleic acid molecules.

There is also provided a method for activating a c-Abl proteincomprising introducing a nucleic acid molecule encoding a c-Abl proteincontaining a protease cleavage site into a cell under conditions inwhich it is expressed and supplying said cell with a protease.

The invention further provides a method for producing an activated c-Ablprotein comprising introducing a nucleic acid molecule encoding a c-Ablprotein containing a protease cleavage site into a cell under conditionsin which it is expressed, supplying the cell with a protease andisolating the cleaved activated C-terminal region of the c-Abl protein.

The invention also provides a method for producing a tyrosine kinaseinhibitor protein according to the aspects of the invention describedabove, comprising introducing a nucleic acid molecule encoding a c-Ablprotein containing a protease cleavage site into a cell under conditionsin which it is expressed, supplying said cell with a protease andisolating the cleaved N-terminal cap region of the c-Abl protein.

The invention further provides a transgenic animal comprising a nucleicacid encoding a c-Abl protein containing a protease cleavage domain, asdescribed above. These transgenic animals are useful as they will enableresearchers to assess the effect of activating the tyrosine kinaseactivity of c-Abl in vivo. According to a further aspect of theinvention, there is provided a method for activating tyrosine kinaseactivity of c-Abl in vivo comprising supplying a transgenic animalcomprising a nucleic acid encoding a c-Abl protein containing a proteasecleavage domain, as described above, with a protease. This method can beconducted using any genetic background in order to assess the effect ofadditional mutations on c-Abl activation. In addition, the transgenicanimal in which the tyrosine kinase activity of c-Abl has been activatedcan be used in in vivo screening assays to identify compounds thatrestore autoinhibition of c-Abl The invention therefore provides an invivo method for screening for a compound that restores autoinhibition ofc-Abl comprising activating the tyrosine kinase activity of c-Abl invivo in a transgenic animal as described above, supplying the transgenicanimal with a candidate compound and assessing the effect of thecandidate compound on the tyrosine kinase activity in the cells of thetransgenic animal. The candidate compound may be any suitable compound.Preferably, the screening method of the invention is used to assess thein vivo effectiveness of modulator compounds identified by the methodsdescribed previously.

Various aspects and embodiments of the present invention will now bedescribed in more detail by way of example, with particular reference tothe inhibition of the tyrosine kinase, Abl. It will appreciated thatmodification of detail may be made without departing from the scope ofthe invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Autoinhibition of c-Abl

A. c-Abl and Abl-PP were expressed by in vitro translation in wheat germextract. Total protein extract (left panel) and anti-Ablimmunoprecipitates (right panel) were separated by SDS-PAGE, blotted tonitrocellulose and probed with anti-Abl and anti-phosphotyrosineantibodies.

B. S. pombe strain SP200 was transformed with a vector expressing c-Abl,ΔSH3-Abl or Abl-PP under control of a thiamine repressable promotor.Sixteen (left panel) and twenty-four hours (right panel) after inductionof protein expression by thiamine removal, cells were lysed by boilingin SDS-sample buffer. Lysates were separated by SDS-PAGE, blotted tonitrocellulose and probed with anti-Abl and anti-phosphotyrosineantibodies.

C. HEK293 cells were transiently transfected with SV40-driven c-Abl andAbl-PP expression plasmids. Forty hours after transfection cells werelysed and Abl protein was immunopurified using covalently coupledanti-Abl antibodies. Abl and co-purifying proteins were eluted by pHshock, separated by 4-15% gradient SDS-PAGE and visualised by colloidalCoomassie staining (right panel). The identity of Abl proteins wasconfirmed by mass spectrometry. Purified c-Abl and Abl-PP proteins wereassayed for their catalytic activity by in vitro kinase assay usingGST-Crk as exogenous substrate. Bands were excised and incorporatedradioactivity was measured by scintillation counting. The histographshows the catalytic activity of purified Abl-PP compared to purifiedc-Abl (mean with SD of two experiments done in triplicate, left panel).Part of the Abl protein used for the in vitro kinase assay was blottedand probed with anti-Abl and anti-phosphotyrosine antibodies (middlepanels). An autoradiograph showing an example of the incorporation ofradioactivity in GST-Crk is presented.

FIG. 2. The core element for c-Abl regulation

A. Schematic diagram representing the different generated Abl proteins.The SH3, SH2 and catalytic domains as well as the last exon region areshown as boxes. P242 and P249 Abl-PP and derivatives.

B. HEK293 cells were transiently transfected with the indicatedSV40-driven Abl expression constructs. Forty hours after transfectioncells were lysed and total protein extract was analysed by anti-Abl andanti-phosphotyrosine immunoblotting.

C. Abl protein was immunoprecipitated from total cell extract usinganti-Abl antibodies, blotted to nitrocellulose and probed with anti-Abland anti-phosphotyrosine antibodies (left panels). Ablimmunoprecipitates were assayed for catalytic activity by in vitrokinase assay using GST-Jun as substrate. The histograph shows thecatalytic activity (mean with SD of three experiments done in duplicate)of the Abl constructs relative to Abl M1-K531.

FIG. 3. Autoinhibition of c-Abl by its N-terminus

A. Schematic diagram representing the different generated Abl proteins.The SH3, SH2 and catalytic domains as well as the last exon region areshown as boxes. P242 and P249 indicate the two proline residues in theSH2-catalytic domain linker that are mutated in Abl-PP and derivatives.

B. HEK293 cells were transiently transfected with c-Abl or the indicatedAbl expression constructs. Forty hours after transfection cells werelysed and the resulting total protein extract was analysed by anti-Abland anti-phosphotyrosine immunoblotting.

C. Abl protein was immunoprecipitated from total cell extract usinganti-Abl antibodies, blotted to nitrocellulose and probed with anti-Abland anti-phosphotyrosine antibodies (left panels). Ablimmunoprecipitates were assayed for catalytic activity by in vitrokinase assay using GST-Crk as substrate. The histograph shows thecatalytic activity (mean with SD of three experiments done in duplicate)of the indicated Abl constructs relative to c-Abl.

FIG. 4. In vitro binding of the N-terminal region to Abl protein

A. c-Abl, Abl ΔM1-D81 or Abl-PP ΔM1-D81 were expressed in HEK293 cells.Cell lysates were incubated with the indicated GST fusion proteins boundto glutathion-Sepharose beads. Adsorbates were analysed by SDS-PAGEfollowed by anti-Abl immunoblotting (left panel). Inputs are shown forAbl (right panel); identical amounts of GST fusion proteins were usedfor each pull-down as quantified by Coomassie blue staining (data notshown).

B. HA-tagged pieces of Abl were expressed in HEK293 cells. Cell lysateswere incubated with the indicated GST fusion proteins as described in A.Adsorbates were analysed by SDS-PAGE followed by anti-HA immunoblotting.

FIG. 5. Mutagenesis analysis of the N-terminal cap

A. Alignment of the c-Abl type 1a and 1b N-terminal regions. Indicatedare the residues (groups of four residues in a row) which are mutated toalanine for the different cap mutants (cap 1-6).

B. Abl cap mutant proteins and the indicated Abl constructs weretransiently expressed in HEK293 cells. Anti-Abl immunoprecipitates wereanalysed by anti-Abl and anti-phosphotyrosine immunoblotting (leftpanels) and assayed for catalytic activity by in vitro kinase assayusing GST-Crk as substrate (right panel). The histograph shows the foldof activation of the different constructs compared to c-Abl (mean withSD of two experiments done in duplicate).

C. HA-tagged Abl protein pieces (SH3-SH2-CD and CD) were expressed inHEK293 cells. Cell lysates were incubated with the indicated GST fusionproteins bound to glutathion-Sepharose beads. Adsorbates were analysedby SDS-PAGE followed by anti-HA immunoblotting.

FIG. 6. Role of the N-terminal cap of c-Abl

A. Schematic diagram showing the structure of the N-terminus of c-Abl.The SH3, SH2 and catalytic domains as well as the last exon region arerepresented by boxes. The amino acid sequence of the N-terminus of thec-Abl type 1b protein is shown. The bold arrow indicates the beginningof the βa strand of the SH3 domain, the engineered TEV cleavage site ismarked in black and the arrowhead indicates the exact position ofcleavage by TEV protease.

B. Abl-TEV and indicated constructs were expressed in HEK293 cells andimmunoprecipitated with anti-Abl antibodies. Immunecomplexes wereincubated without (−) or with (+) TEV protease, separated by SDS-PAGE,blotted to nitrocellulose and probed with anti-Abl antibodies (upperpanel). Immunecomplexes were assayed for catalytic activity by in vitrokinase assay using GST-Crk as substrate (lower panel). The histographshows the fold of activation induced by TEV protease (mean with SD ofthree independent experiments). This was calculated by dividing thecatalytic activity of Abl protein after TEV treatment by the catalyticactivity without TEV incubation.

C. Model of the 3-dimensional arrangement of c-Abl including theN-terminal regulatory “cap” based on known structures of the SH3 domain,the SH12 domain and the catalytic domain of Abl and on regulated Src.The N-terminal “cap” is represented as a rod structure which may bind tothe “north-face” of Abl thus stabilising the regulated structure inwhich the SH3 domain is bound via the SH2-catalytic domain linker to thecatalytic domain. The arrowhead indicates the position of the engineeredTEV cleavage site.

FIG. 7. Inhibition of catalytic activity by the N-terminal cap

A. Abl ΔM1-D81 and Abl-PP ΔM1-D81 were transiently expressed in HEK293cells and immunoprecipitated using anti-Abl antibodies. Immune complexeswere incubated for 2 hours with kinase assay buffer (−) or with equalamounts of GST fusion protein (GST or GST Abl 1b+2) dialysed againstkinase assay buffer, and subsequently assayed for catalytic activity byin vitro kinase assay using GST-Crk as substrate. The histograph showsthe percentage of inhibition (mean with SD of three experiments done induplicate).

B. HEK293 cells were transfected with the indicated constructs. After animmunoprecipitation using anti-Abl antibodies, the immunecomplexes wereanalysed by western-blot anti-Abl (upper panel) or anti-p412 (lowerpanel).

C. Effect of the cap on the catalytic activity of BCR-Abl or ACC BCR-Abltested by in vitro kinase assay using GST-Crk as a substrate. Theactivity of BCR-Abl and ACC BCR-Abl is set to 100%, the histograph showsthe variation of catalytic activity when the cap is restored in BCR-Ablor in ACC BCR-Abl (mean of 2 independent experiments done in duplicatewith SD).

FIG. 8: Alignment of the amino acid sequences of the cap regions forhuman c-Abl 1a and 1b, human Arg 1a and 1b, and murine c-Abl I and IV.The positions of the cap 1 and cap 6 domains are shown.

FIG. 9: Alignment of the nucleic acid sequences of the cap regions forhuman c-Abl 1a and 1b, human Arg 1a and 1b, and murine c-Abl I and IV.

EXAMPLES

1) Results

Expression of c-Abl in Non-Vertebrate Systems

c-Abl and Abl-PP, a deregulated form in which two prolines in theputative intramolecular SH3-binding region connecting the SH2 domain tothe catalytic domain are mutated (P242E/P249E), were expressed in wheatgerm extract. Analysis of total cellular proteins as well as ofimmunoprecitated c-Abl revealed that no tyrosine phosphorylation couldbe detected when c-Abl is expressed (FIG. 1A). However, expression ofAbl-PP resulted in the phosphorylation of c-Abl itself and of a numberof endogenous proteins, showing that c-Abl is regulated in extracts ofplant cells.

We have previously reported that c-Abl and an SH3 domain deletion formof Abl were equally active and toxic in the yeast S. pombe (Walkenhorstet al., 1996). We addressed again regulation of c-Abl in S. pombe, thistime using an inducible promoter of much weaker activity than the oneused previously. In this case, SH3 domain-dependent regulation wasobserved at early induction time points, as judged by a strongdifference in tyrosine phosphorylation of cellular proteins betweenc-Abl and Abl-PP-containing yeast cells (FIG. 1B). After 24 hours,however when more c-Abl protein had accumulated, this difference wasabolished. Thus, c-Abl is regulated in S. pombe, but regulation issensitive to the levels of protein expression.

Regulation of c-Abl In Vitro

We purified c-Abl and Abl-PP from transfected human embryonic kidney(HEK) 293 cells (FIG. 1C). After a purification procedure that includeda high-salt wash step, the Coomassie-blue-stained patterns of c-Abl andAbl-PP proteins were very similar, indicating that any protein(s) thatmay be responsible for the regulation of c-Abl in vivo does notco-purify in detectable amounts or is either too large or too small tobe detected. Constitutively active Abl-PP is strongly phosphorylated ontyrosine and serine residues and thus migrates slightly more slowly thanc-Abl (FIG. 1C, right panel and Dorey et al., 1999). To determine theactivity of the purified Abl proteins we tested their in vitro kinaseactivities using GST-Crk as a substrate. A more than six-fold differencein activity between c-Abl and Abl-PP was measured (FIG. 1C, left panel).We also tested a mutant in which the two prolines in the SH2-CD linkerare mutated to alanine (Abl-P242A/P249A), to find that it was activatedto a similar extent as Abl-P242E/P249E (data not shown). To confirm thatthe difference in activity involved SH3-domain-dependent regulation, wetested various other mutants, including point mutations within the SH3domain, v-Abl and a mutant in which a putative intramolecular saltbridge between the SH3 and catalytic domain is disrupted (Abl K313EBarilá and Superti-Furga, 1998). All deregulated forms were 7 to 16times more active than wild-type c-Abl in in vitro kinase assays (datanot shown). Similar results were also obtained with other anti-Ablantibodies and other substrates (data not shown). These results showthat other cellular proteins are not necessary to maintain c-Abl in astate of catalytic inhibition.

Although it is unclear why it has been difficult to detect suchdifferences previously (Pendergast et al., 1991; Mayer and Baltimore,1994; Dorey et al., 1999) and several parameters may exert an influenceon c-Abl regulation in vitro, we found that the nature of the divalentsalt used in the kinase activity reaction has a profound effect on thecatalytic activity. While the use of MgCl₂ at 10 mM concentration allowsthe detection of significant differences in catalytic activity of c-Ablversus Abl PP, the same samples assayed in the presence of 10 mM MnCl₂resulted in no detectable difference in catalytic activity (data notshown).

Minimal Region Required for c-Abl Regulation

The results on c-Abl regulation in vitro prompted an investigation ofthe minimal part of the protein required for regulation. The “last exonregion” has previously been implicated in c-Abl regulation andrepresents the binding site for several cellular proteins, including F-and G-actin (Goga et al., 1993; McWhirter and Wang, 1993; Van Etten etal., 1994; Woodring et al., 2001). We constructed a deletion mutantlacking the last exon region of c-Abl (Abl M1-K531, FIG. 2A). We alsoengineered a mutant lacking, additionally, the N-terminal amino acidspreceding the SH3 domain, known to be dispensable for regulation of Srcfamily kinases (Abl P82-K531, FIG. 2A).

After transient expression in HEK293 cells, cellular proteins wereanalyzed for phosphotyrosine content as a measure of in vivo proteinactivity and Abl proteins were immunoprecipitated to test for tyrosinephosphorylation as well as catalytic activity in vitro. Deletion of thelast exon region (Abl M1-K531) did not lead to activation of Abl (FIG.2B-C), but the additional double proline mutation in the SH2-catalyticdomain linker (Abl-PP M1-K531) resulted in strong activation. Theseresults suggest that the SH3-domain-dependent regulation is operationalwithin the short form of Abl. Deletion of the last exon region abolishedthe ability of Abl to phosphorylate GST-Crk, but not GST-Jun, probablydue to the loss of the Crk binding site (Ren et al., 1994).

Abl P82-K531 showed elevated tyrosine phosphorylation levels in totalextracts and was highly active in vitro after immunoprecipitation whencompared to its counterpart containing the normal N-terminal sequences(Abl M1-K531; FIG. 2). This suggests an involvement of the first 81residues of the c-Abl protein in regulation which is in sharp contrastto Src family kinases, where residues N-terminal to the SH3 domain aredispensable for intramolecular regulation (Koegl et al., 1995; Sicheriand Kuriyan, 1997). Taken together, our results demonstrate that theminimal region necessary and sufficient for the regulation of c-Ablcomprises the SH3, SH2, catalytic domain as well as the N-terminalresidues.

Regulation of c-Abl by its N-Terminus

To obtain functional insight into the role of the N-terminal region inthe regulation of full-length c-Abl, we constructed additional mutantsand analyzed their catalytic activity in vitro and in vivo (FIG. 3). Asexpected from the results with the short Src-like form (Abl P82-K531),Abl ΔM1-D81 was strongly active also in the presence of the last exonregion and lead to efficient phosphorylation of cellular proteins, Ablautophosphorylation and high catalytic activity in an in vitro kinaseassay (FIG. 3B-C). Introduction of the deregulating mutation in theSH2-catalytic domain linker in Abl ΔM1-D81 (Abl-PP ΔM1-D81) had only asmall further effect, suggesting that through deletion of the N-terminal81 residues, Abl becomes fully deregulated. The effect of individuallydeleting the first 45 residues (exon 1b, Abl ΔM1-H45), or the following36 residues (Abl AE46-D81) was less pronounced (FIG. 3B-C). Thus, theN-terminal amino acids encoded by the Abl type 1b exon and the firstpart of exon 2 are both required to keep c-Abl in its regulated state.

We also tested if the absence of this regulatory N-terminal regionactivated the oncogenic potential of c-Abl. In focus-formation assaysusing NIH3T3-P cells Abl ΔE46-D81 was a reliable oncogene, reaching atransformation efficiency comparable to point mutations in the SH3domain (Table 1; Barilá and Superti-Furga, 1998).

The N-Terminal Region Interacts with c-Abl in trans

We tested the ability of the N-terminus of Abl to interact with the restof c-Abl. GST fusion proteins containing the first exon (amino acids1-45; GST 1b), first and part of the second exon (amino acids 1-80; GST1b+2) and only second exon of c-Abl type 1b (amino acids 46-80; GST 2)were prepared and used to pull-down transiently expressed c-Abl, AblΔM1-D81 and Abl-PP ΔM1-D81 proteins in extracts derived from transfectedHEK293 cells (FIG. 4A). Since in the ABL1 gene two alternative firstexons are spliced to give rise to c-Abl type 1a and 1b (Shtivelman etal., 1986) we also tested similar constructs of c-Abl type 1a (aminoacids 1-26; GST 1a and 1-61; GST 1a+2). All five Abl GST-fusion proteinswere able to pull down Abl ΔM1-D81, suggesting a direct interactionbetween the Abl N-terminal amino acids and Abl ΔM1-D81 in trans. Incontrast, c-Abl was not bound by the GST-fusion proteins, suggestingthat the binding site in c-Abl is “covered” by the N-terminus in cis.Abl-PP ΔM1-D81 bound the GST-fusion proteins only weakly. Thus, theN-terminal region appears to bind the rest of Abl in aconformation-dependent manner.

To define the region in c-Abl to which the N-terminal region binds, GSTfusion proteins of the first exon type 1a, type 1b and of the secondexon were tested for their ability to pull down transiently expressedc-Abl proteins (FIG. 4B), including the SH3-SH2-linker-catalytic domainportion (SH3-SH2-CD), the SH3-SH2 domains or of the catalytic domainonly (CD). The SH3-SH2-CD protein was bound by all three GST fusionproteins. The catalytic domain was bound by first exons 1a and 1b only,while the second exon bound preferentially to the portion of Ablcontaining of the SH3 and SH2 domains. Thus, different parts of theN-terminal region appears to bind different parts of c-Abl, as ifclamping the protein together. Because of its position at the N-terminusof c-Abl, we refer to this region as the “cap”.

Mutational Analysis of the N-Terminal Region

To understand better the nature of the interaction between the “cap” andthe rest of Abl, we performed alanine-scanning mutagenesis of groups offour, mostly polar, residues, in the common second exon region and thatappeared conserved between the 1a and 1b alternative exons (FIG. 5A, cap1-6). After transient expression in HEK293 cells, Abl proteins wereimmunoprecipitated to test for tyrosine phosphorylation content as wellas for catalytic activity in vitro. The cap 1 and cap 6 mutants werephosphorylated and exhibited an increased catalytic activity (FIG. 5B).To establish a possible correlation between the role of cap 1 and cap 6residues in regulation and their binding properties, GST fusion proteinswere prepared of Abl exon 1b with the cap 1 mutation (GST 1b cap 1) andexon 2 with the cap 6 mutation (GST 2 cap 6) and used to pull downtransiently expressed SH3-SH2-CD and CD Abl proteins from HEK293 cellextracts (FIG. 5C). Both mutation of cap 1 and cap 6 residues preventedbinding to the SH3-SH2-CD protein, and the relatively strong binding ofexon 1b to the catalytic domain was strongly reduced by mutation of cap1 residues. These results show that both conserved residues of exon 1and residues of exon 2 are required for the binding to Abl and itsregulation.

The N-Terminal Region is Required to Maintain the Regulated State

The N-terminal region may only be required to assemble the regulatoryapparatus, possibly during folding, and then become dispensable.Alternatively, the N-terminal region may target c-Abl to particularsubcellular sites and only indirectly affect c-Abl regulation. To ruleout these possibilities, we took advantage of the presence of residuesresembling the cleavage site for the highly specific Tobacco-Etch-Virus(TEV) protease roughly at the boundary between the N-terminus and theSH3 domain. We engineered a perfect TEV site by mutating four residuesto obtain Abl-TEV. The TEV cleavage would occur seven residues upstreamof Phe85, representing the beginning of the PA strand of the SH3 domain(FIG. 6A; Musacchio et al., 1994). Mutation of the four residuesrequired to engineer the TEV site did not affect c-Abl regulation (FIG.6B). We transfected HEK293 cells with Abl-TEV as well as with controlAbl constructs. Abl proteins were immunoprecipitated and treated or notwith TEV protease. TEV treatment caused the appearance of afaster-migrating form of Abl-TEV but not of c-Abl (FIG. 6B). Kinaseassays performed in parallel revealed a TEV-dependent increase incatalytic activity only in Abl-TEV and not in c-Abl or Abl ΔM1-D81 (FIG.6B). The extensive washes of the immunoprecipitated protein likelyremoved the cleaved N-terminal portion. Thus, cleaving the N-terminal 77residues of c-Abl in vitro leads to its activation. We conclude that theN-terminal residues are necessary to maintain the inhibited state ofc-Abl and that they have a critical role in its regulation.

The N-Terminal Region Inhibits Abl In Vitro and Restores Regulation ofBCR-ABL

To test whether the interactions of the “cap” are sufficient to restoreAbl regulation in vitro, we purified a GST fusion protein of theN-terminal region of type 1b Abl (GST 1b+2) and incubated it withimmunoprecipitated Abl ΔM1-D81 and with its counterpart bearing theadditional PP mutation in the linker (FIG. 7A). While the GST controlprotein had no effect on catalytic activity, GST 1b+2 inhibited AblΔM1-D81 activity roughly 40%. A significant but reduced level ofinhibition was achieved also with Abl-PP ΔM1-D81. This result couldindicate a potential direct effect on Abl's catalytic activity, ratherthan on mere “regulation” and could also reflect the residual binding ofGST 1b+2 to Abl-PP ΔM1-D81 observed previously (FIG. 4).

BCR-ABL fusion proteins invariably miss the first exon sequences. IfAbl's “cap” region is so powerful that it can re-regulate even activeforms of Abl (such as Abl ΔM1-D81), the missing first exon may restoreregulation of BCR-ABL if reintroduced in the molecule between the BCRsequences and the beginning of the second exon (BCR-cap-Abl; FIG. 7B).In parallel to such constructs, we also tested versions in which thecoiled-coil region of BCR-ABL, critically involved in dimerization, aremissing (ΔCC BCR-cap-Abl; McWhirter et al., 1993). Expression in HEK293cells was equally efficient for all BCR-ABL forms (data not shown).Immunoprecipitation was less efficient for BCR-ABL proteins than for theforms bearing the deletion of the coiled-coil region (FIG. 7B, upperpanel). To monitor BCR-Abl's activity in the cell, we chose to useantibodies specific for tyrosine 412 in the activation loop, likely toreflect the state of activity better than anti-phosphotyrosine, sinceBCR-ABL is tyrosine phosphorylated also at other sites that may not bedirectly dependent on catalytic activity (Pendergast et al., 1993a;Pendergast et al., 1993b). As expected, if compared to the levels ofimmunoprecipitated protein, the forms bearing the coiled-coil deletionwere less phosphorylated at Tyr412, reflecting the importance ofdimerization for BCR-ABL activity (FIG. 7B; McWhirter et al., 1993;Smith and Van Etten, 2001). Introduction of the first exon sequences didnot have a measurable effect on Tyr412 phosphorylation of BCR-ABL butcaused a dramatic reduction in the activity of BCR-Abl bearing thecoiled-coil deletion. To test whether this decrease in activation loopphosphorylation also reflected a reduced catalytic activity in vitro, weperformed kinase assays with the different immunoprecipitated BCR-Ablproteins. The cap-bearing version of ACC BCR-Abl showed about 25% of theactivity of its normal counterpart (FIG. 7C). Together, these data showthat the first exon region is capable of restoring regulation to adimerization-deficient BCR-Abl, confirm the dominant features ofdimerization in overriding the natural regulation of Abl, and suggestthat loss of the first exon may contribute to deregulation of BCR-ABL.

2) Discussion

The data presented here demonstrate regulation of purified c-Abl invitro as well as regulation in non-animal expression systems. Regulationof c-Abl activity is thus an intrinsic property and does not require aparticular cellular inhibitor. An intramolecular sandwich involving theSH3 domain, the linker between the SH2 and catalytic domain and thecatalytic domain itself has been proposed to regulate the activity ofc-Abl as it does in Src family kinases (Barilá and Superti-Furga, 1998).In Src family kinases, however, the assistance of the C-terminal “tail”region is essential. What structure in c-Abl substitutes for theC-terminal tail of Src? The last exon region of c-Abl bears nuclearimport and nuclear export signals on top of binding sites for a varietyof cellular proteins including actin, Crk, Nck, p53. There is geneticevidence that it plays a role in the regulation of c-Abl in cells (Gogaet al., 1993; Woodring et al., 2001). We show however, that the lastexon region of c-Abl is totally dispensable for the SH3 domain-dependentregulation of catalytic activity as defined here and measured in vitro.

If the last exon region is dispensable, what is minimally required? Inaddition to the SH3, SH2 and catalytic domains, the Src-like “core”, wefound an unexpected critical role for the first 81 residues of the c-Ablprotein. In this respect, c-Abl differs to Src family kinases, whereresidues N-terminal to the SH3 domain are dispensable for intramolecularregulation (Koegl et al., 1995; Sicheri and Kuriyan, 1997). An immediatesuggestion concerning a regulatory role of the N-terminus comes from thefact that in the ABL1 gene, two alternative first exons are spliced togive rise to c-Abl type 1a or 1b (Shtivelman et al., 1986). c-Abl 1a is19 amino acids shorter, is spliced much less frequently and does notinclude a myristoylation signal. There is no evidence in the literaturethat would indicate different levels of activity between the two forms.Transgenes encoding both type I and type IV c-Abl proteins rescue thelethality of c-abl mutant mice (Hardin et al., 1996). Our attempts toexpress Abl bearing the type 1a exon in cells failed, consistent withthe report of others (Van Etten, 1999), but when translated inreticulocyte lysates, type 1a Abl is regulated as well as type 1b (K.Dorey, unpublished results). Early reports had addressed the role of theN-terminus in the regulation of c-Abl (Franz et al., 1989; Jackson andBaltimore, 1989; Wang, 1988), but the results were not sufficientlyconclusive, and attention focused on the discovery and function of theadjacent SH3 domain, obscuring the role of the extreme N-terminus ofc-Abl.

We suggest a novel model according to which c-Abl is regulated by a setof intramolecular interactions (FIG. 6C). While Src family kinases havean interaction of their “tail” with their own SH2 domain thatcontributes critically to maintenance of the SH3 domain-dependentregulation, c-Abl has an N-terminal “cap” that serves an analogousfunction. This cap appears to bind at several portions “across” themolecule and stabilize the regulated, inhibited conformation. Accordingto our data, the “KV/LV/LG” motif (see cap 1 region in FIG. 5A), whichis common to both type 1a and 1b exons and required for binding, mustundergo relatively strong interactions with the catalytic domain. Thefirst part of the common second exon does not interact with thecatalytic domain but interacts with the SH3 and/or SH2 domains. FIG. 6Cshows the cap as if binding to the “north-face” merely for graphicconvenience. In fact, because the two regions known to be critical forbinding (the cap 1 and cap 6 residues) are spaced differently in the 1aand 1b forms of Abl, some of the 19 additional residues of the type 1bcap may “loop out” from whatever is the minimal “bridge” from the SH3 tothe catalytic domain.

The experiment with the engineered form of c-Abl in which the presenceof the N-terminal region is removed in vitro, has shown that theN-terminus is required to maintain and not merely to assemble theregulated conformation. This is also confirmed by the ability of capsequences to inhibit cap-less Abl in vitro. This mechanism may beexploited by cellular proteins that inhibit c-Abl in trans.

In general, cellular proteins may either inhibit or activate c-Abl byfavoring or displacing any of the several critical intramolecularinteractions. For example numerous proteins bind the Abl SH3 domain(reviewed in Van Etten, 1999). Moreover, an interdependence of theintramolecular interactions and catalytic activity, as in Src familykinases, seems highly probable (Gonfloni et al., 2000). In this view,phosphorylation of the activation loop with its conformational effectson the catalytic domain and the SH3 and cap-mediated inhibitoryintramolecular interactions antagonize each other. The degree ofcatalytic activity and the degree of SH3 domain availability are the netresult of these opposing forces. In fact, the PTP-PEST tyrosinephosphatase, dephosphorylating the activation loop, acts as an inhibitorof c-Abl (Cong et al., 2000), while Src family kinases and Abl itselfact as activators by causing phosphorylation (Plattner et al., 1999;Brasher and Van Etten, 2000; Dorey et al., 2001).

We believe that the cap represents the missing link in c-Abl'sintramolecular regulation. The first exon region is lacking in all ofthe different fusion proteins formed with BCR or TEL resulting fromchromosomal translocations and also in v-Abl. The dimerizationproperties of BCR and TEL are thought to induce cross-phosphorylationand activation of the catalytic domains by induced proximity and thusrepresent critical “gain-of-function” alterations of c-Abl (Golub etal., 1996; McWhirter et al., 1993; Smith and Van Etten, 2001). Moreover,signaling properties in the BCR portion of BCR-ABL are known to becritical for transformation (reviewed in Sawyers, 1992). Our datasuggest that the absence of the N-terminal cap in BCR-Abl (and inTEL-Abl and v-Abl), represents a “loss-of-function” alteration whichcontributes to the acquisition of constitutive tyrosine kinase activityin these oncogenic forms. Thus, the cap of c-Abl may represent what theC-terminal tail represents for c-Src. A crystal structure of c-Ablincluding the cap will be essential to elucidate the precise molecularmechanism of regulation, and future work will address how the cap maymodulate c-Abl differentially in the different splice variants and inthe context of cellular signaling networks.

3) Experimental Procedures

DNA Constructs

pSGT vector and pSGT-Abl constructs were previously described (Bariláand Superti-Furga, 1998). Abl M1-K531, Abl-PP M1-K531 and Abl P82-K531were obtained by PCR with c-Abl type 1b as template and subcloned inpSGT vector. For preparation of Abl ΔM1-H45, Abl AE46-D81, Abl ΔM1-D81and Abl-PP ΔM1-D81, pSGT-c-Abl or pSGT-Abl-PP were digested with EcoRIand KpnI and the released N-terminal fragment was replaced by a PCRproduct containing the desired deletion. Point mutations and cap mutants(cap 1-6) were obtained using the quick-change site directed mutagenesiskit (Stratagene) and pSGT-c-Abl type 1b DNA as template. All mutagenesisconstructs were confirmed by sequencing. The SH3-SH2-CD (N80-K531),SH3-SH2 (N80-P235) or CD (D252-K531) Abl protein parts were amplifiedusing bAbl as a template and subcloned into a CMV driven vectorcontaining a HA tag (described in Barilá et al., 2000).

The p210 BCR-Abl is a kind gift from Owen Witte. pSGT BCR-Abl and pSGT□CC BCR-Abl have been reconstituted by amplifying respectively M1-F1059and L61-F1059. The resulting BCR-Abl fragments were digested EcoRI/Kpn I(a unique internal site in Abl) and subdloned into pSGT hAbl backbone.To generate pSGT BCR-cap-Abl and pSGT ΔCC BCR-cap-Abl, BCR (M1-S927) orΔCC BCR (L61-S927) have been amplified using p210 as a template. The PCRproducts were digested Eco RI/Not I and subcloned into pSGT hAbl. AllPCR products were sequenced.

Expression of Abl in Wheat Germ Extract and S. pombe

c-Abl and Abl-PP RNA were prepared as described (Dorey et al., 1999) andused for expression of protein in wheat germ extract using a commercialtranslation system (Promega). pSGT-c-Abl, ASH3-Abl and Abl-PP (Bariláand Superti-Furga, 1998) were subcloned to the yeast pRWP vector, aderivative of pRSP (Superti-Furga et al., 1993) containing a mutation ofthe nmt1 promoter making it approximately ten times weaker (Basi et al.,1993), and transformed to S. pombe strain SP200 (Superti-Furga et al.,1993). Expression of Abl protein was induced by removal of thiamine asdescribed (Walkenhorst et al., 1996) and yeast cellular protein extractswere made by boiling pelleted yeast cells in SDS-sample buffer 16 and 24hours after induction.

Transfection and Immunoprecipitation

HEK293 cells were cultured in Dulbecco's Modified Eagle's Mediumsupplemented with 10% fetal calf serum. Cells were transfected withpSGT-Abl DNAs using the calcium phosphate method. Forty hours aftertransfection cells were lysed in IP buffer (50 mM Tris-HCl pH 7.5, 150mM NaCl, 1% NP-40, 5 mM EDTA, 5 mM EGTA, 25 mM NaF, 1 mM orthovanadate,1 mM PMSF, 10 μg/ml TPCK, 5 μg/ml TLCK, 1 μg/ml leupeptin, 1 μg/mlaprotinin, 10 μg/ml soybean trypsin inhibitor) and insoluble materialwas removed by centrifugation (10′ 13,000 rpm). Abl protein wasimmunoprecipitated from total cell lysates in IP buffer using anti-Ablantibody (Ab-3, Oncogene Science or K12, Santa Cruz). Immune complexeswere recovered using protein G-Sepharose beads, assayed for Ablcatalytic activity and analyzed by SDS-PAGE followed by anti-Abl (Ab-3or K12) and anti-phosphotyrosine (4G10, Upstate Biotechnology)immunoblotting. Anti-p412 antibodies that recognize specifically Ablwhen phosphorylated in the activation loop were a kind gift from Dr J.Wu (Cell Signaling Technology (CST) Inc, Beverly, Mass.).

Large-Scale Purification of Abl Protein

c-Abl and Abl-PP protein were expressed by transient transfection inHEK293 cells. Forty hours after transfection cells were lysed in IPbuffer and insoluble material was removed by centrifugation at 100.000gfor 1 h. Cell lysates were incubated with anti-Abl antibodies (Ab-3,Oncogene Science) covalently coupled to protein G-Sepharose beads. Beadswere washed with IP buffer followed by phosphate buffer (50 mMNaPhosphate pH 6.3, 0.1% Triton-X100, 500 mM NaCl). Bound proteins wereused for Abl kinase assay directly or eluted by pH shock (50 mM glycinepH 2.5, 0.1% Triton-X100, 150 mM NaCl) and run on a 4-15% gradientSDS-PAGE gel (Biorad). Abl and co-purifying proteins were detected bybrilliant blue colloidal Coomassie staining (Sigma).

Abl Kinase Assay

The catalytic activity of purified Abl protein bound to proteinG-Sepharose beads was determined as follows. Beads were washed threetimes with IP buffer, two times with IP buffer without NaCl and twotimes with kinase assay buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mMDTT). Twenty μl linase assay mix (1 μg GST-Crk121-226 (Dorey et al.,1999), 0.5 μl [γ-³²P]-ATP (Amersham), 0.1 mM ATP in kinase assay buffer)was added and the mixture was incubated at room temperature for 10 and20 minutes. The kinase reaction was stopped by adding SDS-sample bufferand analyzed by SDS-PAGE. Quantification of the reaction was done bycutting the desired bands from gel followed by liquid scintillationcounting and/or phosphoimager analysis. For the in vitro inhibition ofAbl, GST or GST 1b+2 protein were bacterially expressed, purified anddialysed against kinase assay buffer. Equal quantities of purifiedproteins were added directly to the kinase assay mixture, samples wereincubated for 2 hours at 4° C. and then assayed as above.

Focus Formation Assay

Abl DNAs were subcloned from pSGT to pSLX vector (Renshaw et al., 1995)by BamHI digestion. Focus formation assay was performed in NIH3T3-Pcells (Renshaw et al., 1992) using 0.5 μg of pSLX-Abl DNA essentially asdescribed (Barila and Superti-Furga, 1998; Renshaw et al., 1995).Percentage of foci was calculated by dividing the number of foci by thenumber of neomycin resistant colonies of each construct.

In Vitro Binding Assay

DNA fragments corresponding to c-Abl amino acids, M1-H45 (1st exon type1b), E46-N80 (beginning of the 2nd exon until the start of the SH3domain) and M1-N80 (1 st exon type 1b+2nd exon) were amplified by PCRusing c-Abl type 1b as template. M1-E26 (1st exon type 1a) and M1-N61(1st exon type 1a+2nd exon) were generated using c-Abl type 1a astemplate. All PCR products were cloned in pGEX-2T vector. Bacteriallyproduced Abl GST fusion proteins were pre-bound to glutathion-Sepharosebeads and incubated with 2 mg of HEK293 cell lysate containing c-Abl,Abl ΔM1-D81, Abl-PP ΔM1-D81 or HA-tagged Abl protein parts (SH3-SH2-CD,SH3-SH2 or CD) for 3 hours at 4° C. Bound proteins were analyzed bySDS-PAGE followed by anti-Abl or anti-HA (12CA5, Boehringer Mannheim)immunoblotting.

TEV Cleavage of Abl Protein

An artificial TEV cleavage site was engineered in c-Abl just before theSH3 domain using the quick-change site directed mutagenesis kit(Stratagene). c-Abl amino acids 74-77 (LAGP) were replaced by YFQGintroducing a perfect TEV consensus site, giving rise to Abl-TEV. Afterexpression of Abl-TEV in HEK293 cells and immunoprecipitation usinganti-Abl antibodies (Ab-3, Oncogene Science), the immune complexes boundto protein G-Sepharose beads were incubated for 2 hours at 16° C. in 10mM Tris pH 8.0, 100 mM NaCl, 0.1% Igepal, 0.5 mM EDTA with or without 10units of TEV enme (Gibco). Subsequently, beads were processed asdescribed for Abl kinase assay. TABLE 1 Transformation potential of AblΔE46-D81 Neo colonies Foci in 5% CS Transformation (mean ± SD) (mean ±SD) efficiency (%) PSLX c-Abl 5955 ± 505 13 ± 3 0.2 PSLX Abl ΔE46-D812170 ± 170 215 ± 35 9.9 PSLX Abl-PP 2135 ± 185  615 ± 125 28.8

NIH3T3-P cells transfected with the indicated Abl constructs in vectorpSLX were grown for 16 days in DMEM containing 5% calf serum to allowthe formation of foci. Duplicate dishes were grown under neomycinselection to determine the transfection efficiency. The transformationefficiency was calculated by dividing the number of foci by the numberof neomycin resistant colonies of each construct. Results shown are theaverages of two experiments done in duplicate.

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1. A tyrosine kinase inhibitor protein consisting of the cap region of ac-Abl protein or a functional equivalent thereof.
 2. A tyrosine kinaseinhibitor protein according to claim 1 wherein the c-Abl protein is amammalian c-Abl protein.
 3. A tyrosine kinase inhibitor proteinaccording to claim 2 wherein the c-Abl protein is human.
 4. A tyrosinekinase inhibitor protein according to claim 3 wherein the c-Abl proteinis type 1a c-Abl.
 5. A tyrosine kinase inhibitor protein according toclaim 3 wherein the c-Abl protein is type 1b c-Abl.
 6. A tyrosine kinaseinhibitor protein according to claim 4 consisting of amino acids 1-61 oftype 1a c-Abl.
 7. A tyrosine kinase inhibitor protein according to claim5 consisting of amino acids 1-80 of Type 1b c-Abl.
 8. A tyrosine kinaseinhibitor protein according to claim 2 wherein the c-Abl protein ismurine.
 9. A tyrosine kinase inhibitor protein according to claim 8wherein the c-Abl protein is type 1c-Abl.
 10. A tyrosine kinaseinhibitor protein according to claim 8 wherein the c-Abl protein is typeIV c-Abl.
 11. A tyrosine kinase inhibitor protein according to claim 9consisting of amino acids 1-63 of type I c-Abl.
 12. A tyrosine kinaseinhibitor protein according to claim 10 consisting of amino acids 1-80of type IV c-Abl.
 13. A tyrosine kinase inhibitor protein or functionalequivalent thereof according to claim 1 which inhibits a tyrosine kinaseprotein containing SH2 and SH3 domains.
 14. A tyrosine kinase inhibitorprotein or functional equivalent according to claim 13 which inhibitsAbl, Src or Fyn.
 15. A tyrosine kinase inhibitor protein or functionalequivalent thereof according to claim 13 which inhibits an oncogenicform of said tyrosine kinase protein containing SH2 and SH3 domains. 16.A tyrosine kinase inhibitor protein or functional equivalent thereofaccording to claim 15 which inhibits an oncogenic form of Abl, Src orFyn.
 17. A tyrosine kinase inhibitor protein or functional equivalentthereof according to claim 16 which inhibits an oncogenic form of Abl.18. A tyrosine kinase inhibitor protein or functional equivalent thereofaccording to claim 17 which inhibits BCR-Abl.
 19. A fusion proteincomprising a tyrosine kinase inhibitor protein or a functionalequivalent thereof according to claim 1 fused to a marker domain.
 20. Afusion protein according to claim 19 wherein the marker domain is greenfluorescent protein.
 21. An antibody that which binds to a tyrosinekinase inhibitor protein or a functional equivalent thereof according toclaim
 1. 22. A nucleic acid molecule encoding a tyrosine kinaseinhibitor protein or a functional equivalent thereof according toclaim
 1. 23. An antisense nucleic acid molecule which binds under highstringency conditions to a nucleic acid molecule according to claim 22.24. A vector comprising a nucleic acid molecule according to claim 22.25. A host cell transformed or transfected with a nucleic acid moleculeaccording to claim
 22. 26. A method for preparing a tyrosine kinaseinhibitor protein or a functional equivalent thereof comprisingculturing a host cell containing a nucleic acid molecule according toclaim 22 under conditions whereby said protein is expressed andrecovering said protein thus produced.
 27. A method of identifying anactivator compounds that inhibits autoinhibition of c-Abl by the capregion comprising contacting c-Abl with a candidate activator compoundand assessing whether binding between the cap region of c-Abl and thecatalytic and SH2 and/or SH3 domains of c-Abl has been inhibited.
 28. Anactivator compound identified or identifiable by the method of claim 27.29. A method for identifying a modulator compound that restoresautoinhibition of c-Abl by the cap region comprising contacting c-Abland an activator compound according to claim 28 with a candidatemodulator compound and assessing whether binding between the cap regionof c-Abl and the catalytic and SH2 and/or SH3 domains of c-Abl isrestored.
 30. A modulator compound identified or identifiable by themethod of claim
 29. 31. A method of modulating the activity of a proteintyrosine kinase comprising providing a cell with an active agent saidactive agent selected from the group consisting of (i) a tyrosine kinaseinhibitor protein or a functional equivalent thereof according to claim(ii) a fusion protein comprising said tyrosine kinase inhibitor proteinor a functional equivalent thereof fused to a maker domain; (iii) anucleic acid molecule encoding said tyrosine kinase inhibitor protein ora functional equivalent thereof; (iv) a nucleic acid molecule encodingsaid fusion protein; (v) an antisense molecule which binds under highstringency conditions to a nucleic acid molecule of (iii); (vi) anantisense molecule which binds under high stringency conditions to anucleic acid molecule of (iv); (vii) an activator compound that inhibitsautoinhibition of c-Abl by the cap region; and (viii) a modulationcompound that restores autoinhibition of c-Abl by the cap region. 32.Use of a cap region of a c-Abl protein or a functional equivalentthereof as a tyrosine kinase inhibitor.
 33. An active agent for use as apharmaceutical said active agent selected from the group consisting of:(i) a tyrosine kinase inhibitor protein or a functional equivalentthereof according to claim 1; (ii) a fusion protein comprising saidtyrosine kinase inhibitor protein or a functional equivalent thereoffused to a maker domain; (iii) a nucleic acid molecule encoding saidtyrosine kinase inhibitor protein or a functional equivalent thereof;(iv) a nucleic acid molecule encoding said fusion protein; (v) anantisense molecule which binds under high stringency conditions to anucleic acid molecule of (iii); (vi) an antisense molecule which bindsunder high stringency conditions to a nucleic acid molecule of (iv);(vii) an activator compound that inhibits autoinhibition of c-Abl by thecap region; and (viii) a modulation compound that restoresautoinhibition of c-Abl by the cap region.
 34. A pharmaceuticalcomposition comprising an active agent in conjunction with apharmaceutically-acceptable carrier molecule, said active agent selectedfrom the group consisting of: (i) a tyrosine kinase inhibitor protein ora functional equivalent thereof according to claim 1; (ii) a fusionprotein comprising said tyrosine kinase inhibitor protein or afunctional equivalent thereof fused to a maker domain; (iii) a nucleicacid molecule encoding said tyrosine kinase inhibitor protein or afunctional equivalent thereof; (iv) a nucleic acid molecule encodingsaid fusion protein; (v) an antisense molecule which binds under highstringency conditions to a nucleic acid molecule of (iii); (vi) anantisense molecule which binds under high stringency conditions to anucleic acid molecule of (iv); (vii) an activator compound that inhibitsautoinhibition of c-Abl by the cap region; and (viii) a modulationcompound that restores autoinhibition of c-Abl by the cap region. 35.Use of an active agent in the manufacture of a medicament for thetreatment of a disease with aberrant tyrosine kinase activity, saidagent selected from the group consisting of: (i) a tyrosine kinaseinhibitor protein or a functional equivalent thereof according to claim1; (ii) a fusion protein comprising said tyrosine kinase inhibitorprotein or a functional equivalent thereof fused to a maker domain;(iii) a nucleic acid molecule encoding said tyrosine kinase inhibitorprotein or a functional equivalent thereof; (iv) a nucleic acid moleculeencoding said fusion protein; (v) an antisense molecule which bindsunder high stringency conditions to a nucleic acid molecule of (iii);(vi) an antisense molecule which binds under high stringency conditionsto a nucleic acid molecule of (iv); (vii) an activator compound thatinhibits autoinhibition of c-Abl by the cap region; and (viii) amodulation compound that restores autoinhibition of c-Abl by the capregion.
 36. A method of treating a disease associated with aberranttyrosine kinase activity in a patient, comprising administering to thepatient an active agent selected from the group consisting of: (i) atyrosine kinase inhibitor protein or a functional equivalent thereofaccording to claim 1; (ii) a fusion protein comprising said tyrosinekinase inhibitor protein or a functional equivalent thereof fused to amaker domain; (iii) a nucleic acid molecule encoding said tyrosinekinase inhibitor protein or a functional equivalent thereof; (iv) anucleic acid molecule encoding said fusion protein; (v) an antisensemolecule which binds under high stringency conditions to a nucleic acidmolecule of (iii); (vi) an antisense molecule which binds under highstringency conditions to a nucleic acid molecule of (iv); (vii) anactivator compound that inhibits autoinhibition of c-Abl by the capregion; (viii) a modulation compound that restores autoinhibition ofc-Abl by the cap region; and (xi) a pharmaceutical compositioncomprising (ii), (iii), (iv), (v), (vi), (vii) or (viii).
 37. The methodaccording to claim 36 wherein said disease is a neurological disease orcancer.
 38. The method according to claim 37 wherein said disease isleukaemia.
 39. A method of diagnosing a conditions associated with anaberrant activity of a tyrosine kinase protein comprising measuring thelevel of an aberrant tyrosine kinase protein in a cell sample obtainedfrom a patient using a tyrosine kinase inhibitor protein according toclaim
 1. 40. A method of diagnosing a condition associated with anaberrant tyrosine kinase activity of c-Abl protein comprising using anucleic acid molecule according to claim 22 to screen for mutations inthe cap region.
 41. A transgenic animal comprising a nucleic acidmolecule according to claim
 22. 42. A c-Abl protein comprising aprotease cleavage site located near the boundary of the cap region andthe SH3 domain.
 43. A c-Abl protein according to claim 42 wherein saidprotease cleavage site is a TEV protease cleavage site.
 44. A fusionprotein comprising a c-Abl protein according to claim 42 fused to amarker domain.
 45. A method for activating the tyrosine kinase activityof c-Abl comprising supplying a cell with a c-Abl protein comprising aprotease cleavage site according to claim 42 and supplying the cell witha protease to cleave at the protease cleavage site.
 46. A method forproducing an activated c-Abl protein comprising cleaving a c-Abl proteinaccording to claim 42 with a protease and isolating the cleavedC-terminal region of said c-Abl protein.
 47. A method for producing atyrosine kinase inhibitor protein comprising cleaving a c-Abl proteinaccording to claim 42 with a protease and isolating the cleavedN-terminal cap region of said c-Abl protein.
 48. A nucleic acid moleculeencoding a c-Abl protein according to claim
 42. 49. A method foractivating a c-Abl protein comprising introducing a nucleic acidmolecule according to claim 48 into a cell under conditions in which itis expressed and supplying said cell with a protease.
 50. A method forproducing an activated c-Abl protein comprising introducing a nucleicacid molecule according to claim 48 into a cell under conditions inwhich it is expressed, supplying said cell with a protease and isolatingthe cleaved C-terminal region of said c-Abl protein
 51. A method forproducing a tyrosine kinase inhibitor protein comprising introducing anucleic acid molecule according to claim 48 into a cell under conditionsin which it is expressed, supplying said cell with a protease andisolating the cleaved N-terminal cap region of said c-Abl protein.
 52. Atransgenic animal comprising a nucleic acid molecule according to claim48.
 53. A method for activating tyrosine kinase activity of c-Abl invivo comprising supplying a transgenic animal according to claim 52 witha protease.
 54. A method for screening for a compound that restoresautoinhibition of c-Abl in vivo comprising supplying a transgenic animalaccording to claim 52 with a protease to activate the tyrosine kinaseactivity of c-Abl, supplying the transgenic animal with a candidatecompound and assessing the effect of the candidate compound on thetyrosine kinase activity in the cells of said transgenic animal.
 55. Anantibody that binds to a fusion protein according to claim
 19. 56. Anucleic acid molecule encoding a fusion protein according to claim 19.57. An antisense nucleic acid which binds under high stringencyconditions to a nucleic acid molecule according to claim
 56. 58. Avector comprising a nucleic acid molecule according to claim
 23. 59. Avector comprising a nucleic acid molecule according to claim
 56. 60. Avector comprising a nucleic acid molecule according to claim
 57. 61. Ahost cell transformed or transfected with nucleic acid moleculeaccording to claim
 23. 62. A host cell transformed or transfected withnucleic acid molecule according to claim
 56. 63. A host cell transformedor transfected with nucleic acid molecule according to claim
 57. 64. Ahost cell transformed or transfected with a vector according to claim24.
 65. A host cell transformed or transfected with a vector accordingto claim
 58. 66. A host cell transformed or transfected with a vectoraccording to claim
 59. 67. A host cell transformed or transfected with avector according to claim
 60. 68. A method for preparing a tyrosinekinase inhibitor protein or a functional equivalent thereof comprisingculturing a host cell containing a nucleic acid molecule according toclaim 56 under conditions whereby said protein is expressed andrecovering said protein thus produced.
 69. A method of diagnosing acondition associated with an aberrant tyrosine kinase activity of c-Ablprotein comprising using a nucleic acid molecule according to claim 22to screen for mutations in the cap region.
 70. A method for activatingthe tyrosine kinase activity of c-Abl comprising supplying a cell with ac-Abl protein comprising a protease cleavage site according to claim 44or a fusion protein and supplying the cell with a protease to cleave atthe protease cleavage site.
 71. A method for producing an activatedc-Abl protein comprising cleaving a c-Abl protein according to a fusionprotein according to claim 44 with a protease and isolating the cleavedC-terminal region of said c-Abl protein.
 72. A method for producing atyrosine kinase inhibitor protein comprising cleaving a c-Abl proteinaccording to a fusion protein according to claim 44 with a protease andisolating the cleaved N-terminal cap region of said c-Abl protein.
 73. Anucleic acid molecule encoding a c-Abl protein or a fusion proteinaccording to claim 44.